Bottom Line:
Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma.Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes.TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

ABSTRACTAn early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

Figure 5: TLTF targeting signals direct a heterologous reporter protein (GFP) to vesicles that are localized to the trypanosome flagellar pocket. Procyclic T. brucei cells were transiently transfected with a T. brucei expression plasmid containing the GFP gene alone (top) or a plasmid encoding a GFP–TLTF fusion protein (bottom). The transfected cells were examined by laser scanning confocal microscopy and the transmitted and fluorescent images were superimposed on one another. GFP fluorescence is green. Cells in the bottom panel were additionally stained with ethidium bromide to visualize the nucleus and kinetoplast (red staining). The white bar corresponds to 10 μm.

Mentions:
To examine the cellular fate of TLTF, we fused it to GFP from Aquorea victoria (11). Constructs encoding either this fusion protein, or GFP alone, were expressed in transiently transfected trypanosomes, which were examined by laser scanning confocal microscopy (Fig. 5). Because Northern blot analysis indicated that TLTF is expressed in both procyclic and bloodstream form trypanosomes (data not shown), we used the more readily cultured procyclic forms for these transfection experiments. Western blots using an α-GFP polyclonal antiserum (Clonetech, Palo Alto, CA) confirmed that transfected trypanosomes were producing a protein of the expected size (data not shown). Cells in the bottom panel of Fig. 5 were also stained with ethidium bromide to visualize the nucleus and kinetoplast (red staining). As can be seen in the top panel of Fig. 5, GFP alone is expressed evenly throughout the entire cell. The GFP– TLTF fusion protein, on the other hand, is restricted to vesicles that are usually, but not exclusively, located at or near the trypanosome flagellar pocket (Fig. 5, bottom). These results are consistent with TLTF being a secreted protein, as shown previously (15), and demonstrate that signals for TLTF targeting are located within the protein itself, even though it does not contain a hydrophobic NH2-terminal signal sequence. Thus, TLTF is added to the growing list of secreted eukaryotic proteins for which the amino acid sequence gives no indication of a readily identifiable targeting signal (18). Interestingly, many such proteins play a role in immune system function (e.g., IL-1β and adult T cell leukemia-derived factor [ADF]. IL-1β is a monocyte-derived, proinflammatory cytokine that stimulates a broad range of cell types. ADF mediates upregulation of IL-2 receptor expression and may be involved in the abnormal proliferation of T cells observed in some types of leukemogenesis (19). Although some features of export may be shared between leaderless secreted proteins, the mechanisms responsible are unknown. Recent data indicate that multiple mechanisms exist (20) and that two different proteins may utilize partially, but not completely, overlapping pathways (21).

Figure 5: TLTF targeting signals direct a heterologous reporter protein (GFP) to vesicles that are localized to the trypanosome flagellar pocket. Procyclic T. brucei cells were transiently transfected with a T. brucei expression plasmid containing the GFP gene alone (top) or a plasmid encoding a GFP–TLTF fusion protein (bottom). The transfected cells were examined by laser scanning confocal microscopy and the transmitted and fluorescent images were superimposed on one another. GFP fluorescence is green. Cells in the bottom panel were additionally stained with ethidium bromide to visualize the nucleus and kinetoplast (red staining). The white bar corresponds to 10 μm.

Mentions:
To examine the cellular fate of TLTF, we fused it to GFP from Aquorea victoria (11). Constructs encoding either this fusion protein, or GFP alone, were expressed in transiently transfected trypanosomes, which were examined by laser scanning confocal microscopy (Fig. 5). Because Northern blot analysis indicated that TLTF is expressed in both procyclic and bloodstream form trypanosomes (data not shown), we used the more readily cultured procyclic forms for these transfection experiments. Western blots using an α-GFP polyclonal antiserum (Clonetech, Palo Alto, CA) confirmed that transfected trypanosomes were producing a protein of the expected size (data not shown). Cells in the bottom panel of Fig. 5 were also stained with ethidium bromide to visualize the nucleus and kinetoplast (red staining). As can be seen in the top panel of Fig. 5, GFP alone is expressed evenly throughout the entire cell. The GFP– TLTF fusion protein, on the other hand, is restricted to vesicles that are usually, but not exclusively, located at or near the trypanosome flagellar pocket (Fig. 5, bottom). These results are consistent with TLTF being a secreted protein, as shown previously (15), and demonstrate that signals for TLTF targeting are located within the protein itself, even though it does not contain a hydrophobic NH2-terminal signal sequence. Thus, TLTF is added to the growing list of secreted eukaryotic proteins for which the amino acid sequence gives no indication of a readily identifiable targeting signal (18). Interestingly, many such proteins play a role in immune system function (e.g., IL-1β and adult T cell leukemia-derived factor [ADF]. IL-1β is a monocyte-derived, proinflammatory cytokine that stimulates a broad range of cell types. ADF mediates upregulation of IL-2 receptor expression and may be involved in the abnormal proliferation of T cells observed in some types of leukemogenesis (19). Although some features of export may be shared between leaderless secreted proteins, the mechanisms responsible are unknown. Recent data indicate that multiple mechanisms exist (20) and that two different proteins may utilize partially, but not completely, overlapping pathways (21).

Bottom Line:
Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma.Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes.TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

ABSTRACTAn early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.